1. Field of the Invention
This invention is directed to animal feeds used in aquaculture or in agriculture, with microbial cells as components. These microbial cells contain exogenous peptides, proteins, and/or antibodies, which can convey resistance or immunity to pathogens (such as viral or bacterial), or otherwise improve the health and performance of the species that consume them. The microbial cells can be algae, bacteria, fungi, or yeast. The exogenous peptides, proteins, and/or antibodies can be expressed inside the microbial cells by direct genetic modification of the microbe or by infecting the microbe with a virus that has been altered to express the protein of interest. The invention is also directed to animal feed supplements and therapeutics with microbial cells as components.
2. Related Art
Plant products have been produced using specific genetic modification to express proteins and/or antibodies of therapeutic value. The group at the Boyce Thompson Institute at Cornell has cloned a viral coat protein into bananas capable delivering an oral vaccine when ingested by humans, but this concept has not been extended to microbes.
There are several plant biotech companies, such as Meristem, Large Scale Biology, and Prodigene, which are now expressing certain human therapeutic proteins, including antibodies, in plants. Large Scale Biology is expressing proteins in tobacco plants using a tobacco mosaic virus as a vector to produce the protein of interest. The protein is then isolated and purified from the plant material and used for human therapeutic purposes. In this way, the plant genome itself is actually not modified, but rather the genome of the infecting virus carries the gene of interest.
Recombinant microbes, including bacteria, yeast, and other fungi, have been used to produce human therapeutic proteins. However, such recombinant microbes have not been used in agriculture or agriculture, wherein the cultivated animal ingests the whole organism. Rather, to date, the recombinant organism has been used as a factory from which the therapeutic protein is isolated and purified prior to use.
Certain plant products have been produced that contain proteins and/or antibodies of therapeutic value. They have been produced by infecting the plant with a virus that expresses the protein of interest. Large Scale Biology has a series of patents protecting this technology, but its purpose is to produce purified proteins for pharmaceutical purposes, which requires an extensive purification procedure following harvesting of the plant material. These patents do not involve the use of the crude plant material as a source of both nutrition and disease control, except under the unusual condition that the pharmaceutical product is expressed in the fruit of the plant.
Certain recombinant proteins have been produced in insect cells using an insect virus expression system (baculovirus). These proteins are also produced in intact insect larvae following infection with modified baculoviruses. In both cases, the insect cells or larvae are used as factories to produce the protein of interest, and the recombinant protein is then purified for pharmaceutical purposes. Insect cells or larvae infected with baculovirus are particularly useful in the expression of certain human therapeutic proteins because the post-translational modifications of the therapeutic proteins are similar to the post-translational modifications imparted upon expression in human cells.
A baculovirus expression system is an efficient method for expressing proteins in insect cell culture. Baculovirus is in the family Baculoviridae, a diverse group of large double stranded DNA viruses that infect arthropods, including insects, arachnids, and crustaceans. Baculoviruses are species-specific and do not infect vertebrates, nor can they propagate in mammalian cells in culture.
The Sindbis arbovirus can be used to deliver high levels of gene expression in vivo in non-host arthropod species without causing cytopathic effects in infected cells or impairing the development of the organism. A replication competent Sindbis virus containing the coding region of green fluorescent protein (GFP) induced productive infections when injected into insect larvae and pupae (Lewis, et al., 1999). Thus, virus-mediated ectopic gene expression has been accomplished in arthropods, a phylum that includes the classes Crustacea and Insecta.
Antibiotic doping is used routinely in the aquaculture setting. Typically, the pure or semipure antibiotics are added directly to the water column. However, crude fermentation broths, or crude preparations including cells, have not been used for any kind of therapeutic delivery system.
Production of amino acids, such as lysine, typically involves a genetically modified microorganism, which overproduces the amino acid of interest and excretes it into the fermentation medium. The wastestream from such a fermentation would include biomass containing the amino acid, and this wastestream product could be used as a crude delivery form of the small molecule nutritive amino acid.
Microalgae (single celled algae or phytoplankton) represent the largest, but most poorly understood, kingdom of microorganisms on the earth. As plants are to terrestrial animals, microalgae represent the natural nutritional base and primary source of all the phytonutrients in the aquatic food chain. As the primary producers in the aquatic food chain, microalgae are the source of many phytonutrients, including docosahexaenoic acid (DHA) and arachidonic acid (ARA). Microalgae also represent a vast genetic resource, comprising in excess of 80,000 different species.
Yeast, filamentous fungi, and bacteria are also in the direct food chain of fish, crustaceans, and mollusks. However, only a very few of these microbes, perhaps less than 10 species, have been exploited for aquaculture feeds. These few species have been used primarily for historical reasons and ease of cultivation. They have not been chosen on the basis of any scientific evidence of superiority as nutritional or therapeutic supplements.
The marine environment is filled with bacteria and viruses that can attack fish and shellfish, thereby devastating aquaculture farms very quickly. Bacteria and viruses can also attack single celled microalgae, so these organisms have evolved biochemical mechanisms to defend themselves from such attacks. Such mechanisms may involve the secretion of probiotic compounds that inhibit bacterial growth or viral attachment.